Low-molecular-weight heparins (“LMWHs”) are an important class of antithrombotic compounds. LMWHs are safe and effective for the prevention and treatment of venous thromboembolism and several are approved for these uses in Europe and North America. The LMWHs have the advantage that they can be given subcutaneously and do not require APTT monitoring. Thus, LMWHs permit the outpatient treatment of conditions such as deep vein thrombosis and pulmonary embolism that previously required hospitalization.
LMWHs are glycosaminoglycans consisting of chains of alternating residues of D-glucosamine and one of the two uronic acids: uronic acid, either glucuronic acid or iduronic acid. While unfractionated heparin is a heterogeneous mixture of polysaccharide chains ranging in molecular weight from about 3,000 to 30,000, LMWHs—which are often produced by a controlled enzymatic or chemical depolymerization of heparin—have mean molecular weights of about 5,000. All heparins derive their anticoagulant activity from activating antithrombin (previously known as antithrombin III). This activation is mediated by a unique pentasaccharide sequence that is randomly distributed along the heparin chains. About one third of the chains of unfractionated heparin, but only 15 to 25 percent of the chains of LMWHs, contain the pentasaccharide sequence.
There are differences between unfractionated heparin and LMWHs relating to their relative inhibitory activity against factor Xa and thrombin. Any pentasaccharide-containing heparin chain can inhibit the action of factor Xa simply by binding to antithrombin. However, to inactivate thrombin, heparin must form a ternary complex, by binding to both antithrombin and thrombin. Such complexes are only formed by pentasaccharide-containing heparin. Since fewer than half of LMWHs molecules are of sufficient length to bind to both antithrombin and thrombin, LMWHs have less activity than that against thrombin. On the other hand, LMWH have greater activity against factor Xa. The relative importance of inhibition of factor Xa and inhibition of thrombin in mediating an antithrombotic effect varies with the clinical setting, but there is evidence that both are necessary. Accordingly, the LMWHs have increasingly been used for the therapy of conditions with an elevated risk of blot clots.
The LMWHs are typically formulated for clinical use in aqueous solutions. Because of their potent anti-coagulant activity it is necessary to accurately monitor LMWHs' concentrations during their formulation and packaging into pharmaceuticals. Further, the ability to measure the concentration of an experimental sample is fundamental to any research and development program seeking to invent new glycosaminoglycan anticoagulants.
Prior to the current invention, such measurements have required the use of UV light absorption and refractive index-based techniques. However, the use of UV absorption is complicated by the fact that some LMWHs produce only a very weak UV signal that is nearly undetectable. In addition, UV methods require large amounts of test material making them impractical, especially when sample volumes are limiting. Further, UV absorption is not readily adaptable to the rapid, high-throughput quantitative assay format. Similarly, refractive index-based techniques lack specificity and can result in artificially high results. Such inaccuracies are particularly undesirable when measuring drug levels.
Thus, there remains a need for methods that can measure the concentration of glycosaminoglycan anticoagulants, such as LMWHs, in solution which are accurately quantitative, quick, easily performed, and amenable to high throughput.